The genome sequence of the European flat oyster, Ostrea edulis (Linnaeus, 1758)

We present a genome assembly from an individual Ostrea edulis (the European flat oyster; Mollusca; Bivalvia; Ostreida; Ostreidae). The genome sequence is 894.8 megabases in span. Most of the assembly is scaffolded into 10 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 16.35 kilobases in length.


Background
Ostrea edulis, the European flat oyster, is a variably round to oval or pear-shaped bivalve that grows to approximately 110 mm in diameter.It has a cream or off-white shell, which is usually discoloured grey or covered in epiphytic growth.It can be found living in the low intertidal and shallow shelf seas on bottoms of firm mud and hard silt, particularly associated with estuarine habitats (Perry & Jackson, 2017).Ecologically, it serves as a keystone species and habitat engineer, forming extensive beds, which are important habitats and feeding grounds for many species, thus greatly increasing biodiversity where it is present (Bennema et al., 2020;Coen et al., 2007;Grabowski & Peterson, 2007).
Ostrea edulis has been the target of reintroduction and restocking projects and conservation efforts (Bromley et al., 2016;Rodriguez-Perez et al., 2019).It is currently listed as threatened or declining by OSPAR (Haelters & Kerckhof, 2009).
The genome of Ostrea edulis has been previously sequenced (Gundappa et al., 2022).In this work, as a part of the Darwin Tree of Life programme, we present a chromosomally-complete sequence of the species, based on a specimen collected from Devon, UK.

Genome sequence report
The genome was sequenced from one Ostrea edulis (Figure 1) collected from Western Point, Oreston, Devon, UK (50.36,.A total of 38-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 94 missing joins or mis-joins and removed 33 haplotypic duplications, reducing the assembly length by 70.95% and the scaffold number by 48.54%, and increasing the scaffold N50 by 0.35%. The final assembly has a total length of 894.8 Mb in 52 sequence scaffolds with a scaffold N50 of 94.3 Mb (Table 1).Most (99.79%) of the assembly sequence was assigned to 10 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-Figure 5; Table 2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/37623.DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The xbOstEdul1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Muscle tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts.High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit.HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30.Sheared DNA was purified by solidphase reversible immobilisation using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample.The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
RNA was extracted from muscle tissue of xbOstEdul1 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.RNA was then eluted in 50 μl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 instruments.Hi-C data were also generated from muscle tissue of xbOstEdul1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023).The assembly was checked for contamination and corrected using the gEVAL system (Chow et al., 2016) as described previously (Howe et al., 2021).Manual curation was performed using gEVAL, HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022).The mitochondrial    (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file Table 3. Software tools: versions and sources.
Table 3 contains a list of relevant software tool versions and sources.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Miguel Naranjo-Ortiz
University of Oslo, Oslo, Norway The methodology is solid and the results are well presented and accessible.
There are two aspects I believe would enhance the paper.These comments are just a suggestion and not a requirement: The first is providing a more comprehensive description of the mitochondrial genome.The mitochondrial genome has been widely studied in mollusks, so it is a point of interest in this group in particular.
The second is a comparison with the previous reference genome for the species.While I understand that this assembly is of a much higher quality, having two independent assemblies can provide a good view into the intraspecific genomic diversity of the species.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Comparative genomics, evolutionary genomics, mycology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.Reviewer Expertise: Oyster genomics, genetics and disease.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 2 .
Figure 2. Genome assembly of Ostrea edulis, xbOstEdul1.1:metrics.The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 894,803,243 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (112,480,954 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (94,306,699 and 75,578,129 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the mollusca_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/xbOstEdul1.1/dataset/CANOQP01/snail.

Figure 3 .
Figure 3. Genome assembly of Ostrea edulis, xbOstEdul1.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/xbOstEdul1.1/dataset/CANOQP01/blob.

Figure 4 .
Figure 4. Genome assembly of Ostrea edulis, xbOstEdul1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/xbOstEdul1.1/dataset/CANOQP01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Ostrea edulis, xbOstEdul1.1:Hi-C contact map of the xbOstEdul1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=ZheqF50kSq23AUjYR99Udw.

Reviewer Report 27
March 2024 https://doi.org/10.21956/wellcomeopenres.22053.r75505© 2024 Bean T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Tim P. Bean The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, UK This paper represents a new high-quality genomic resource for the European flat oyster Ostrea edulis.All the data required is provided and the methods used to generate it are clearly described.This paper and associated genome data will provide an excellent resource for the research and conservation communities.I have just a few minor comments below... Native oysters have highly variable shell colour.Maybe remove reference to colouration.○ "700 million oysters caught in London".Should this say eaten in London?○ I can't find evidence to support the statement that M. gigas has replaced O. edulis (within the cited Haelters paper).Maybe remove this statement.○ Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes Are sufficient details of methods and materials provided to allow replication by others?Yes Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.

Open Peer Review Current Peer Review Status: Version 1
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.